DESIGN OF A MEMBRANE-PROTEIN FOR SITE-SPECIFIC PROTEOLYSIS - PROPERTIES OF ENGINEERED FACTOR XA PROTEASE SITES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Lactose permease is a polytopic membrane transport protein with 12 hyd rophobic transmembrane domains connected by hydrophilic loops on the c ytoplasmic and periplasmic sides of the membrane. By the use of an act ive permease mutant devoid of Cys residues (C-less permease), single r ecognition sites (Ile-Glu-Gly-Arg) for the protease factor Xa (fXa) we re engineered into hydrophilic loops 7, 8, and 10 in the C-terminal ha lf of the protein. Mutants carrying single sites inserted at position 255, 259 (loop 7), 283, 286 (loop 8), or 341 (loop 10) exhibit signifi cant lactose accumulation (30-70% of C-less permease) and normal level s of expression in the membrane. However, despite solubilization in do decyl beta-D-maltoside, none of the mutant permeases is proteolyzed by fXa to a significant extent. Insertion of two recognition sites in ta ndem at position 255 results in partial cleavage, and remarkably, intr oduction of three sites in tandem leads to complete proteolysis by fXa . Importantly, mutants with two or three fXa sites at position 255 acc umulate lactose to high levels (70% of C-less) and are present in the membrane in amounts comparable to that of C-less permease. The results indicate that hydrophilic loops 7, 8, and 10 are buried in the tertia ry structure of the permease where they are inaccessible to protease. Insertion of tandem sites probably facilitates proteolysis by causing loops to become more accessible to the aqueous phase and by increasing the local concentration of protease recognition sites. The approach s hould be applicable to other polytopic membrane proteins.